RESEARCH ARTICLE Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads Huy Quoc Nguyen1‡, Jagoree Roy2†, Bjo¨ rn Harink1†, Nikhil P Damle2†§, Naomi R Latorraca3, Brian C Baxter4, Kara Brower5, Scott A Longwell5, Tanja Kortemme6,7, Kurt S Thorn4#, Martha S Cyert2, Polly Morrell Fordyce1,5,7,8* 1Department of Genetics, Stanford University, Stanford, United States; 2Department of Biology, Stanford University, Stanford, United States; 3Biophysics Program, Stanford University, Stanford, United States; 4Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States; 5Department of Bioengineering, Stanford University, Stanford, United States; 6Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, United States; 7Chan Zuckerberg Biohub, San Francisco, United States; 8ChEM-H Institute, Stanford University, Stanford, United States *For correspondence: Abstract Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) [email protected] in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of † These authors contributed these interactions is essential for deciphering and perturbing signaling networks but is challenging equally to this work due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously Present address: ‡Genentech, quantifies protein binding to a library of peptides directly synthesized on beads containing unique South San Francisco, United spectral codes. Using MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved States; §OSTHUS GmbH, protein phosphatase essential for the immune response and target of immunosuppressants, to the Eisenbahnweg, Aachen, PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically # Germany; Zymergen, Inc, contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with Emeryville, United States a wide range of affinities. The quantitative biophysical data provided by this approach will improve Competing interests: The computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and authors declare that no revolutionize our understanding of signaling networks. competing interests exist. DOI: https://doi.org/10.7554/eLife.40499.001 Funding: See page 23 Received: 08 August 2018 Accepted: 03 July 2019 Published: 08 July 2019 Introduction In vivo, rapid regulation of weak, transient protein-protein interactions is essential for dynamically Reviewing editor: Michael A shaping cellular responses. Nearly 40% of these interactions are mediated by 3–10 amino acid Short Marletta, University of California, Linear Motifs (SLiMs) interacting with protein globular domains (e.g. SH3, SH2, and PDZ domains) or Berkeley, United States enzymes (e.g. kinases and phosphatases) (Dinkel et al., 2016; Neduva and Russell, 2006; Copyright Nguyen et al. This Tompa et al., 2014). The human proteome is estimated to contain more than 100,000 of these article is distributed under the SLiMs, many of which are highly regulated by post-translational modifications (PTMs) such as phos- terms of the Creative Commons phorylation (Tompa et al., 2014; Ivarsson and Jemth, 2019). As the weak affinities of these interac- Attribution License, which tions (K values of ~1 to 500 mM) are often close to the physiological concentrations of the permits unrestricted use and d redistribution provided that the interacting partners in vivo, subtle differences in concentrations or interaction affinities can have original author and source are large effects on the fraction bound and downstream signaling output (Roy et al., 2007; Hein et al., credited. 2017). Measuring and predicting not only which SLiMs a protein binds but also the affinities of Nguyen et al. eLife 2019;8:e40499. DOI: https://doi.org/10.7554/eLife.40499 1 of 28 Research article Biochemistry and Chemical Biology Structural Biology and Molecular Biophysics SLiM-binding interactions is therefore essential for predicting signal strengths within signaling net- works, understanding how these networks are perturbed by human disease, and identifying new therapeutic inhibitors (Uyar et al., 2014). Calcineurin (CN), a conserved Ca2+/calmodulin-dependent phosphatase, is a prime example of a signaling protein that relies on weak SLiM-mediated interactions for substrate recognition. Although CN plays critical roles in the human immune, nervous, and cardiovascular systems and likely dephos- phorylates hundreds of downstream targets, only ~70 are known to date (Sheftic et al., 2016). These include the NFAT family of transcription factors, whose dephosphorylation by CN is required for T-cell activation and adaptive immunity (Jain et al., 1993). Consequently, CN is the target of the widely used immunosuppressants cyclosporin A (CysA) and FK506. CN dephosphorylates sites with little sequence similarity, instead recognizing substrates by binding to two characterized SLiMs (PxIxIT and LxVP) located at variable distances from the phosphosite (Roy and Cyert, 2009). Block- ing SLiM binding to CN prevents dephosphorylation without altering its catalytic center: FK506 and CysA prevent LxVP docking, the viral inhibitor A238L blocks PxIxIT and LxVP binding, and the high- affinity peptide inhibitor PVIVIT blocks PxIxIT binding (Grigoriu et al., 2013; Aramburu et al., 1999). The affinities of PxIxIT motifs determine biological output by specifying the Ca2+ concentra- tion-dependence of substrate dephosphorylation in vivo (Mu¨ller et al., 2009; Roy et al., 2007). However, the relationship between PxIxIT sequence and CN binding affinity has never been probed outside of the core motif. A comprehensive understanding of PxIxIT-CN binding would allow discov- ery of novel CN substrates and aid efforts to rationally design CN inhibitors with enhanced selectivity. Unfortunately, the limited binding interfaces associated with SLiM-mediated interactions result in low to moderate affinities (with typical Kd values in the range of 1–500 uM), high dissociation rates (Zhou, 2012; Dogan et al., 2015), and a rapid equilibrium (Gianni and Jemth, 2017; Bag- shaw, 2017), complicating experimental efforts to measure affinities. Display-based methods such as combinatorial phage display (Tonikian et al., 2008) and ProP-PD (Ivarsson et al., 2014; Sundell and Ivarsson, 2014) allow screening for binding between a protein of interest and large libraries (up to 1010) of candidate SLiM-containing peptides. However, these methods typically iden- tify only strong binders, cannot return negative information about residues that ablate binding criti- cal for downstream target prediction in vivo (Gfeller et al., 2011), and do not directly probe effects of PTMs. Array-based methods (Fodor et al., 1991) allow quantification of binding of labeled pro- teins to 10s-100s (SPOT arrays) (Frank et al., 1990), tens of thousands (Atwater et al., 2018), or mil- lions (ultra-high density arrays) (Buus et al., 2012; Forsstro¨m et al., 2014; Price et al., 2012; Carmona et al., 2015) of peptides chemically synthesized in situ, permitting direct incorporation of PTMs or unnatural amino acids at specific positions (Engelmann et al., 2014; Tinti et al., 2013; Filippakopoulos et al., 2012). However, it is difficult to evaluate the yield and purity of peptides in each spot, contributing to false positives and negatives (Tinti et al., 2013; Blikstad and Ivarsson, 2015). Most importantly, display- and array-based screening methods cannot measure quantitative interaction affinities (Kds or DDGs), as these measurements require washes that take the system out of thermodynamic equilibrium and lead to preferential loss of interactions with faster off-rates (Zhou, 2012; Dogan et al., 2015; Ivarsson and Jemth, 2019). As a result, candidate interactions identified through screening methods are typically validated and their affinities quantified via subse- quent quantitative, low-throughput methods that require large amounts of material and are labor- intensive (e.g. isothermal calorimetry, fluorescence polarization, or surface plasmon resonance) (Gibson et al., 2015; Davey et al., 2017; Dinkel et al., 2016; Tonikian et al., 2008). The ability to measure binding affinities for hundreds of protein-peptide interactions in parallel would simulta- neously remove this validation and quantification bottleneck and allow quantitative mapping of bind- ing specificity landscapes to improve target prediction in vivo. Here, we present a powerful bead-based technology for quantitatively measuring affinities for many SLiM-mediated protein-peptide interactions in parallel using very small amounts of material. Peptides are synthesized directly on spectrally encoded beads (MRBLEs, Microspheres with Ratio- metric Barcode Lanthanide Encoding) (Gerver et al., 2012; Nguyen et al., 2017a) with a unique linkage between each peptide sequence and a given spectral code. MRBLE-pep libraries can then be pooled and assayed for protein binding in a single small volume before being imaged to identify the peptide sequence associated with each bead and quantify the amount of protein bound. On- MRBLE chemical synthesis allows for precise control of peptide density, incorporation of PTMs at Nguyen et al. eLife 2019;8:e40499. DOI: https://doi.org/10.7554/eLife.40499 2 of 28 Research article Biochemistry and Chemical Biology Structural